Fig 2.

Analysis of the binding dynamics of NS5A-GFP to ER and LD membranes. (A) Dual FRAP analysis of NS5A-GFP (green) and GRASP65-DiHcRed (red) in Huh7 cells. A rectangle over an area containing a cluster of LDs in a cell coexpressing NS5A-GFP and DiHcRed-GRASP65 was photobleached using high-power Ar 488-nm and He Ne 543-nm lasers. Images were captured for approximately 4 min. (B) Quantitative analysis of the experiment whose results are shown in panel A. The average fluorescence intensity of NS5A-GFP (green filled circles) and DiHcRed-GRASP65 (red filled circles) in the bleach box is plotted against time and fitted to the equation described in Materials and Methods. τ values are the inverse of the k of the exponential. R² values were 0.92 and 0.99 for NS5A-GFP and GRASP65, respectively. (C) FRAP beam-size analysis of ER membrane-associated NS5A-GFP. Typical FRAP curves of NS5A-GFP using ×63 and ×40 objectives at 37°C. Solid lines, best fit of a nonlinear regression analysis (Materials and Methods). Note the different time scales between the two objectives. (D) FRAP beam-size analysis. Bars are the mean ± standard error of the mean of 35 to 45 measurements. The τ values (left) and ratios (right) are shown. (E) Comparison between the ER and LD binding of NS5A using saponin treatment. COS7 cells expressing NS5A-GFP (green and bottom) were labeled with Bodipy (red) after 4 h of OA treatment. Saponin was added to a final concentration of 0.2%, and images were captured for several minutes. (Inset) Magnified NS5A-GFP-decorated LDs. (F) Quantitative analysis of the experiment described in panel E. The changes in relative residual fluorescence intensity of NS5A-GFP in two regions of interest over LDs and the ER are plotted against time upon addition of the detergent. Data were fitted to an exponential-decay equation. The results shown are typical results from three independent experiments.